Method for producing therapeutic product



Nov. 20, 1951 v. MENKIN METHOD FOR PRODUCING THERAPEUTIC PRODUCTS Filed May 2l, 1949 5 Sheets-Sheet l 30,0 ern afa/7 a//d/ yze unf/l Free of 504 On /'c For' one o Jevera/ days l or euko c fos/Is -pranvong Facfor (LPF) ma@ INVENToR.

Nov. 20, 1951 v. MENKIN METHOD FOR PROOUOING THERAPEUTIC PRODUCTS Filed May 21. 1949 5 Sheets-Sheet 2 Nov. 20, 1951 v. MENKIN METHOD FCR PRODUCING THERAPEUTIC PRODUCTS Filed May 2l, 1949 5 Sheets-Sheet 3 Nov. 20, 1951 v. MENKIN 2,575,763

METHOD FOR PRODUCING THERAPEUTIC PRODUCTS Filed May 2l. 1949 5 Sheets-Sheet 4 )K JNVENTOR.

BY l W may Nov. 20, 1951 v. MENKIN METHOD FOR PRODUCING THERAPEUTIC PRODUCTS Filed May 2l, 1949 5 Sheets-Sheet 5 INVENTUR.

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Patented Nov. 20, l1951 UNITED STATES PATENT ,OFFICE METHOD FOR PRODUCING THERAPEUTIC PRODUCT Valy Menkin, Philadelphia, Pa.

Application May 21, 1949, Serial No. 94,689

1 Claim.

This isla continuation-impart of my copending application, Serial No. 714,667 led on Dccember 6, 1946, now abandoned.

This invention relates to a leukocytosis-promoting factor (LPF) obtained from inflammatory exudates derived from various animal forms,

including man, and to processes for the prepara-A tion of the same.

It has been known for many years that nucleic acids and their hydrolytic products have an influence on the total white blood cell count of animals. Among the materials which are known to produce leukocytosis, resulting in an increase in the polymorphonuclear leukocytes, are

nucleic'acids, yeast, nucleic acids from thymus, l'

locytosis is a syndrome characterized by a marked decrease in white cells due especially to the This disease is serious and the mortality rate is high.. Hence it is obvious that an agent which will promote,Y leukocytosis (the manufacture of white blood cells) is highly desirable as a therapeutic agent for the treatment of agranulocytosis. Also, it is well yknown that prognosis of a number ofpatholog'ic conditions is to a large extent referable to the number of circulating leukocytes. lt is, therefore, apparent that the administration of, aLPF will prove to be clinically valuable in the `treatment of many abnormal conditions whereinV the white blood cell content has been reduced, as in anemias such as aplastic anemia. or severe leukopenia.

While pentose nucleotide, which contains the sodium salts of` four nucleotides from the ribo` nucleic acid of yeast, and the other materials mentioned above, have been useful in the treatment .of diseases involving a reduction in the 'number of leukocytes, all of these therapeutic agents have many disadvantages. Among these disadvantages are side reactions encountered from lirn',ran1uscular injection of pentose nucleotides. Also effects on the patients following administration, such as chills, fever, a tight sensation in the chest, a feeling of tension, and inner-excitement followed by lethargy, semistupor, and dullness present many difculties. .f In .accordance 4 with the present invention, these Adisadvantages are overcome byV aleukocy-` tesis-promoting factor obtained from inammatory exudates. This factor is essentially a thermolabile, non-dialyzable 2-globulin-associ ated fraction obtained from the inflammatory exudates which primarily exerts an effecten the hematopoietic tissue in the bone marrow `to cause a rise in the number of immature polymorphonuclear leukocytes, said leukocytosis-prometing factor being essentially free from substances having leukopenic activity.

In isolating the leukocytosis-promoting factor from inflammatory exudates, the exudate is treated with a salt, preferably ammonium sulfate, and the precipitate `containing undesirable face tors from a leukocytosis-promoting standpoint is discarded. Further salt is `then added to the remaining Isupernatant liquid in greater concena tration, andthe supernatant liquid resulting from further salting out is centrifuged to obtain the leukocytosis-promoting factor.

The object of my invention and the invention itself will be understoodby reference to the the invention.

EXAMPLE Pleural exudation was induced by the injection, under ether anesthesia, of 1.5 cc. of turpentine into the right vchest of dogs. Severalhours following the injection of the irritant, a sample of the exudate was withdrawn by means `of a Luer syringewith a hypodermic needle. The latter was of large caliber and filed off at the end in order to diminish the chance of injury to the lungs.

To the sample of this inflammatory exudate in a beaker is added ammonium sulfate to onethird saturation. The resulting precipitate 'is discarded and the supernatant liquid is dialyzed until free of sulfate ions.' Ammonium sulfate is Vthen added to one-halfv saturation and the Whole is cooled to below' 5 C. by 'remaining in' the refrigerator for severaldays. A precipitate sediments. The 'clear supernatant `phase -i's then syphoned 01T or decanted. The sediment which remains is centrifuged. The supernatant phase remains cloudy. This phase is then dialyzed. This material, which has been dialyzed, contains the leukocytosis-promoting factor, is then dried by Dry Ice and desiccated further in vacuo over phosphoric anhydride. The activity of purified LPF can be prolonged by maintaining the active material in tlie presence of ammonium sulfate on ice. The sulfate ions are dialyzed out prior to use of he material. In this way the LPF can be maintained for weeks in the iiuid state on ice. After dialysis, drying by freezing can be employed. Average yield 12.8 milligrams per cc. of exudate.

The pharmacological results of injecting the above material into dogs is describedin my publication The Active Principle in the Leukocytosis- Promoting Factor of Y Exudates (Blood, The Journal of Hematologyivol. III, No. 8, August Apparently when the LPF' obtained -by the above process is aged spontaneous denaturation occurs and a relatively inactive and insoluble part is formed. The active principle is split off in the form of a soluble component.

It is clear that when 10 to 20 milligrams of aged LPF.l (3 6 months old) is treated with about 10 cc. of saline, stirred, and centrifuged, the supernatant part yields considerable activity when injected into dogs. There is an increase of about 64 percent in the number ofV circulating leukocytes. The data of several such experiments appears in the following Table 1.

TABLE 1 Eect of drsoluble fraction. derived from aged LPF (3-6 months old) on the leukocyte level Mximlfimlnump er o w lite fitffg Basal nur nblood cells Dog No. 1 which soluble ber ofwhite within -holrs fraction derived -blood cells following ad-` ministration of material Mg. Cu. mm Ou. mm. 3-T 10 18, 850 30, 250 {1l-T 14. 5 19, 000 46, 125 B-D 14 11,000 27, 175 5-T- 13. 5 16, 975 24, 050 G-T, 14 19, 000 22, 250 8-T- 11.5 16, 950 27, 300 8-T. 17 18, 700 20, 100 9-T.-. 10 16,000 25, 700 10-T.. 20 9, 800 16, 850 ll-T 14, 700 24, 450

Average.. 16, 097 l 26, 425

l Percentage increasel in leukocyte level=64.2%.

The evidence indicates that the active principle is liberated in'toto as a soluble component from the new insoluble and aged sample of leukocytoss-promoting factor. (See Figure `2 of drawings.)

The LPF was extracted from exudates of dogs, as described above. Various quantities ofthe factor in the fluid state were treated with crystalline trypsin in amounts varying from a mere pinch of the enzyme to 2 milligrams. The length of incubation with the -LPF was also variable, lasting from about one hour to over twelve hours. The treated LPF failed to be inactivated by tryptic digestion. The observations are assembled in Table 2. It is quite'clear -that the addition of trypsin has Ifailed to inactivate the factor. Following Vsuch digestion the injection of the treated material still-caused a rise of 111.5

percent' in the number of circulating leukocytes 4 (Table 2). The course of an experiment is graphically shown in Figure 3 of the drawings.

TABLE 2 E'lect of tryptic digestion on the activity of the leukocytosz's-promoting factor Maximum number of white Basal num- .blood cells Dog No. Material injected Whbrgod Wglllllgh'frs cells jection o1 treated LPF with enzyme Cu. mm. Cu. mm. 11-T. 13 cc. LPF-l-pinch crystal- 12,100 24, 600 line trypsin 12-T 18 cc. LPF' cubated over- 7, 225 11,300

night with trypsin. l6-T 20 cc. LPF incubated with 12, 275 25. 900

trypsin 2 hours. 9-T 18 cc. LPF incubated with 10, 200 30. 900

trypsin 2 hours. 17-T. 17 0c. LPF incubated 2 6,300 p 8, 950

hours with 2 mgm. trypsin. 20-T l0 cc. LPF incubated l 10. 900 24,100

hour and 5 minutes with 1 mgm. crystalline trypsin. 0-1 10 cc. LPF incubated 1 l0, 750 2l. 750

hour and 25 minutes with approx. 1 mgm. crystalline trypsin.

Average 9, 964 1 21, 071

l Percentage increase in leukocyte levcl=1l1.5%.

When the newly obtained leukocytosis-promoting lfactor is heated for 30-35 minutes atV 100 C., the whole molecule appears to be denatured and the LPF is likewise inactivated. The results ofV these experiments are summarized in Table 3 below.

TABLE, 3

Eect of heat (at C'. for 30-35 minutes) on the activity of the Zeukocytosz's-promotz'ng factor I Maximumv I t BasailS white cell wl-itslel-cell colnt Amoun oi coun in experi- 3 ours fo Expenment No' LPF used meut with unlowing adminisheated LPF tration of un heated L PF Mg. Cu. mm Cu. mm 20 8, 500 17, 400 19 9, 425 ,500 17 l0. 575 16. 550 25 11, 800 27. 950 23 l1. 000 Y 23, 250

Average 10, 260 20, 930

Maximum Basal white W4hi6tel:2 cell crulnt Amount of count in experiours c Experiment No' LPF used ment with lowing adminis- Y heated LPF stration of heated LPF Mg. Cu. mm Cu. mm. Y

20 9. 375 9, 550 19 7` 350 8, 350 17 s. 825 s. 40o Y 25 9, 425 10. 600 23 13. 275 14. 150

Average 9, 65o ro. 210

Percentage increase in leukocyte level with unheated LPF=104%. Percentage increase inleukocyte level with heated,LPF=6%.

4er thedrawings.)

When on the contrary, the active supernatant o1' soluble fraction obtained from aged LPF is evaporated to dryness on a steam bath, its activity remains essentially intact. Evaporation of the active supernatant material over a steam bath fails to inactivate the principle. The results of these experiments are collected together yin Table 4 below.

TABLE 4 Effect of the soluble fraction derived from aged LPF when evaporated to dryness on steam bath and also when that dried fraction is boiled for 30-40 minutes Maximum whlitelcell count o owlng lngg administration natant material of elther wapo" Dog No derived from Basal White rated material aged LPF cell counts dervdpflrom age l or megtefgnto following boilY ing o such evaporated dried material Mg. Ca. mm Cn. mm. 9-T 7, 625 17, 500 11, O50 24, 200 9. 350 20. 650 12, 950 18. 400

Average. 10. 244 1 20,188

Average. 13, 275 3 2e. 500

l Percentage increase in leukocyte level=97.1%.

2 The evaporated material to dryness has in addition been subjected to boiling for 30-40 minutes.

a Percentage increase in leukocyte level=1l4.7%.

When such brittle, dried material obtained by evaporation over a steam bath is heated for 30-40 minutes at 100 C., the active principle fails to be inactivated. (See Table 4.) The active supernatant material wh-en evaporated to dryness forms brittle ilakes which are insoluble in an aqueous medium. Heating again to 100 C. such insoluble material fails to decrease its potency. Its injection in dogs induces a rise of 114.7 percent in the level of circulating leukocytes. This observation definitely indicates that the LPF can be recovered from aged exudates as a highly thermostable substance. The results of these experiments appear in Figure 5 of the drawings.

Polypeptides are known to be highly thermostable. For this reason the amino acid nitrogen before and after hydrolysis was determined on several samples of the active supernatant phase from an aged sample of LPF. The measurements of such samples indicate in each case a rise in the amino acid nitrogen following acid hydrolysis. The figures obtained on such samples before and after hydrolysis are listed in Table 5 below. These observations suggest very strongly that the active principle is a relatively simple polypeptide.

TABLE 5 The amino nitrogen content in the active principle of the LPF before and after hydrolysis The example given above is intended to illustrate the invention without limiting it to the exact materials, proportions, and conditions described therein. In the example given, two fractionations of the exudate are made to obtain the LPF. By the use of this procedure optimum yields of LPF are obtained. However, it is to be understood that LPF can also be obtained by procedures which involve further fractionations.

The LPF, when injected into man causes an increase in the number of circulating leukocytes extending over a period of many hours. Results of these experiments are shown in a paper published in the Archives of Pathology; April 1946 (page 379 and elsewhere throughout the article).

What I claim is:

A process for the isolation of a leukocytosispromoting factor from inflammatory animal exudates which comprises fractionating the exudates with ammonium sulfate to one-third saturation, discarding the resulting precipitate, dialyzing the resulting supernatant liquid until free of sulfate ions, adding ammonium sulfate to one-half saturation, cooling, siphoning off the albumin which is a clear supernatant phase and collecting the unsettled part of the precipitate, oentrifuging the supernatant containing unsettled precipitate and discarding the precipitate which settles, dialyzing the unsettled precipitate until free of sulfate ions, and drying the dialyzed material in vacuo.

VALY MENKIN.

REFERENCES CITED The following references are of record in the le of this patent:

UNITED STATES PATENTS Number Name Date 2,161,861 Gerlough June 13, 1939 2,246,355 Gerlough June 17, 1941 OTHER REFERENCES Menkin in Am. J. Path. 19, pp. 1021-1027, Nov. 1943.

Am. J. Med. Sci. 205, Dp. 363-368, Mar. 1943.

Proc. Soc. Exptl. Biol. Med. 56, pp. 219-220, June 1944. 

